Download effect of arsenic stress on amino acid profile

EFFECT OF ARSENIC STRESS ON AMINO
ACID PROFILE
4.1. Material and Methods
4.1.1. Amino acid profiling
The pico tag kit from Waters (Milford, MA, USA) was used for estimation of
amino acids on Waters – HPLC system (Bidlingmeyer et al., 1984). 0.2 g of
homogenized rice plant samples were hydrolysed in 10 ml of 6 N HCl in an oven for 12
hours at 120ºC. 10 µl of filtered hydrolysed samples (Flow chart) and of standard (2.5
µmoles/ml in 0.1 N HCl) were derivatised with phenylisothiocynate (PITC) in vaccum
oven at 55ºC for 30 mins at 75 milli torr after three rounds of drying and redrying. The
derivatised samples were then diluted with pico tag sample diluent and filtered with
syringe filters. 20 µl of this was then injected into the system. Chromatographic analysis
of the extracts was performed with a Waters Binary gradient HPLC system with
accessories module 2475, (Waters, Milford, MA, USA) equipped with a degasser (DG2),
a binary pump module (515), Temperature control module (TC2), Pump control module
(PC2) and a photodiode array detector (Waters 2998). The separation was carried out at
40 °C using a Pico Tag amino acid C18 column (3.9 X 15 cm; 5 µm). For each sample,
20 µl of extract was injected and the column was eluted at 1 ml min-1, with an optimized
gradient established using solvents A (0.14 M sodium acetate, containing 0.05%
triethylamine and 6% acetonitrile, pH 6.40) and B (60% acetonitrile in water). A step-bystep gradient was used with an increase of proportion of solvent B until it reached 46%
during 10 min, followed by an increase upto 100% in 5 min, with a flux of 1 ml min -1.
The column was then cleared and optimised to 100% A for 8 min at 1 ml min-1.
The amino acids analysed were Aspartic acid (Asp), Glutamic acid (Glu), Serine
(Ser), Glycine (Gly), Histidine (His), Arginine (Arg), Threonine (Thr), Alanine (Ala),
Proline (Pro), Tyrosine (Tyr), Valine (Val), Methionine (Met), Cysteine (Cys),
Isoleucine (Ile), Leucine (Leu), Phenalanine (Phe) and Lysine (Lys). Tryptophan (Trp),
Asparagine (Asn) and Glutamine (Gln) could not be analysed by this procedure as these
are heat labile. Chromatograms were integrated using Empower 2 HPLC software v6.0.
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Effect of Arsenic Stress on Amino Acid Profile
HYDROLYS IS PROCED URE
0.2—0.3 grams of ho mogenized Plant
fish tissue (15 –20% of Protein) + 10 mL of 6 N HcL + Phenol Crystal
(0.5 mg ) + Nit rogen Flush + Heat Seal
20—24 hrs. Hydrolysis at 105°C
(or)
One hour Hydrolysis at 150°C (low yields for Serine, threonine and tyrosine)
REDRYING PROCEDURE
5 µL of filtered Hydrolysis sample / 5 µL of Protein Hydrolysate Std
(2.5 µmo les / mL in 0.1 N HcL)
Vacuum Dry at 55 °C for 30 minutes at 75 milli torr
2 X 10 µL of Red rying Solution
& Vortex for a few seconds to mix
(2 : 2:1 )
(0.5 : 0.5 : 0.25) Water : Ethanol : Triethylamine
Vacuum Dry at 55 °C for 30 minutes at 75 milli torr
DERIVATIZATION PROCED URE
10 µL of derivatizat ion reagent
& Vortex for a few seconds to mix
(7:1:1:1 )
(210 :30 :30 :30)
(Ethanol : Triethylamine: Water:PITC)
Vacuum Dry at 55 °C for 30 minutes at 75 milli torr
Makeup to 1 mL with Pico Tag Sample Diluent
4.1.2 Estimation of Free Proline and Cysteine
Extraction and determination of free proline was performed according to the
method of Bates et al. (1973). Plant samples (1g) were extracted with 3% Sulphosalicylic
acid. Extracts (2ml) were held for 1h in boiling water by adding 2ml Ninhydrin and 2ml
glacial acetic acid, after which cold toluene (4ml) was added. Proline content was
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Effect of Arsenic Stress on Amino Acid Profile
measured by a spectrophotometer (Biorad Shimadzu UV 1601) at 520 nm and calculated
as µmol g-1 fw against standard proline.
Free cysteine content of As treated and untreated rice shoots/roots was measured
according to the method by Gaitonde (1967) with the help of spectorphotometer (Biorad
Shimadzu UV 1601). Plant material (500 mg) was homogenized in 5% chilled perchloric
acid and centrifuged at 10,000 × g for 10 min at 4ºC. Cysteine content was measured in
supernatant using acid-ninhydrin reagent. For preparation of every 10 ml of acid
ninhydrin reagent, 250 mg of ninhydrin was dissolved in 6 ml glacial acetic acid and 4
ml HCl. Reaction mixture (3 ml) contained one ml each of supernatant, glacial acetic
acid and acid ninhydrin reagent. Mixture was heated for 15 min at 95ºC, and then cooled
rapidly to room temperature and absorbance was recorded at 560 nm. Cysteine content
was calculated from the standard curve prepared using known concentrations of cysteine
(L-cysteine hydrochloride, Sigma) and is expressed as µmol g-1 fw.
4.2 Results
4.2.1. Amino acid profile
Amino acid profiling of root and shoot (Fig. 4.1, 4.2) parts of the two contrasting
genotypes was undertaken. In general, the content of different amino acids showed
significant decline in HARG upon exposures to higher concentration of As(III) than
As(V) as compared to control.
The As accumulation was differentially correlated to different amino acid content
in the two contrasting genotypes. The total amino acid content was significantly
correlated to As in LARG (R=0.965***), while it was negatively correlated in HARG
(R= -0.796**). Serine, threonine, tyrosine, arginine and valine were found negatively
correlated to As accumulations in both the genotypes. While, some amino acids leucine,
aspartic acid, histidine, isoleucine and lysine were significantly negatively correlated to
As accumulation in HARG and not in LARG. Cysteine content in plant significantly
correlated with As accumulation in LARG roots (R= 0.953***) and shoots (R= 0.937**)
(Appendix C-D). While, in HARG it was significantly correlated in shoots (R=0.993**)
but not in roots (R=0.573NS). In this study glutamic acid (Glu) as well as glycine (Gly)
were significantly correlated with As accumulation in LARG shoots (Glu; R=0.945***,
Gly: R=0.902**). Glutamic acid was not linearly correlated with As accumulations
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Effect of Arsenic Stress on Amino Acid Profile
(R=0.236) in roots of HARG. On the other hand, the induction in Gly synthesis was
linearly correlated in both the genotypes upon AsIII and AsV exposure. Proline content
was especially significantly correlated to As accumulations in LARG shoots and roots
(R=0.919*** and R=0.924** respectively), while in HARG though the content was more
but non-linearly correlated to As accumulations.
Alanine increased at increasing exposures most significantly in LARG shoots at
25 µM As(III) (166%) (Fig. 4.1). Arginine decreased with increasing As exposures, more
in roots of both genotypes upto 87% and 88% at 10 µM As(V) in LARG and HARG
(Fig. 4.1). Aspartic acid increased in LARG (43% at 25 µM As(III) in shoots) but
decreased in HARG (93% at 25 µM As(III) in shoots). Cysteine increased in both
genotypes. Most significantly in LARG roots (by 524% at 25 µM As(III)) (Fig. 4.1).
Glutamic acid increased in both the genotypes being most in HARG roots (959% at 50
µM As(V) (Fig. 4.1). Glycine increased in both the genotypes but most significantly in
HARG shoots (239% at 25 µM As(III)). Histidine increased in LARG and decreased in
HARG. The contrast was most in shoots exposed to higher concentrations of both As(III)
and As(V). Isoleucine increased in LARG (most in roots at 25 µM As(III) exposures)
and decreased in HARG (by 92% at 10 µM As(V)) (Fig. 4.1). Leucine on the other hand
showed significant decrease in HARG (by 99% in shoots at 25 µM As(V)), while in
LARG insignificant induction was also seen (20% at 50 µM As(V) in shoots) (Fig. 4.1).
Significant decrease in Lys was seen in HARG roots (by 77% at 50 µM As(V))
while in LARG no specific trend was seen with decrease in shoots upto 31% in As(III)
10 µM and increase by 59% in roots at 25 µM As(III) exposures (Fig. 4.2). Methionine
increased with increasing exposures in both the genotypes being most significant in
HARG root (by 361%) at 50 µM As(V) (Fig. 4.2). Respsonse of Phe to As exposures
was variable in the two genotypes with increase in LARG shoots (by 78% at 25 µM
As(III) and decrease in LARG roots, HARG shoots and HARG roots (most in HARG
roots at 10 µM As(V)) (Fig. 4.2). Proline was induced in both the genotypes at all
exposures from 107% (in HARG roots at 25 µM As(III)) to 387% (in LARG shoots at 25
µM As(III)) (Fig. 4.2). Serine decreased in both the genotypes with increasing exposures
being most in HARG shoots (91% at 25 µM As(III)) (Fig. 4.2). Threonine decreased
with increasing exposures in HARG (by 75% in roots at 25 µM As(III)) and increase by
60% in LARG shoots exposed to 50 µM As(V)) (Fig. 4.2). Increase in Tyr was observed
only in roots of LARG (upto 90% at 10 µM As(V)) but decrease in shoots was by 70% at
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Effect of Arsenic Stress on Amino Acid Profile
25 µM As(III). In HARG roots and shoots the decrease was by 91% at 25 µM As(III) in
roots (Fig. 4.2).
Cysteine content in plant significantly correlated with As accumulation (R=
0.899*) but, increases in PC-SH showed a nonlinear relationship with free cysteine in
HARG (R= 0.497NS). In this study Glu as well as Gly were significantly correlated with
GSH synthesis (p<0.001 except Gly in HARG; p<0.01). Free to bound ratio of Cys
increased by nearly two times in LARG, while a decrease of up to two times was noticed
in HARG (Fig. 4.4). The increase in total Cys level was 15 times more in LARG
(R=0.908*) root than in HARG at 25µM As(III) (R=0.621NS). Glutamic acid increased
by nearly two times (R=0.965**) in root and shoot of LARG, while in HARG it
increased by three times as compared to control. On the other hand, the induction in Gly
synthesis was 13 fold in LARG and 7 fold in HARG root as compared to control (Fig.
4.1 and 4.2).
Total proline was induced by 4 fold in HARG and 2 fold in LARG root. Free to
bound Proline ratio increased two times more in root of HARG than LARG (at 25µM
As(III); Fig. 4.3) corresponding to As accumulation (R=0.845*) (Appendix C-D).
When exposed to 10 µM As(V) Pro increased by 355 % in LARG Shoot & 267
% in HARG Shoot (Fig. 4.3) moreso increasing its free to bound ratio. Pro enhanced by
115 % in LARG Root & by 152 % in HARG Root upon exposure to 10 µM As(V). Pro
showed an increase with increase in As(V) concentration. Pro enhanced by 143 % in
LARG Shoot & by 240% in HARG Shoot at 50 µM concentration of As(V). It was
enhanced by 261 % in LARG Root & by 235 % in HARG Root at the same treatment of
As(V). The metalloid accumulation and free / bound proline accumulation was
significantly correlated in HARG shoots (R= 0.909***) and roots (R=0.864***) upon
As(III) exposures. There was 72 % increment of Cystein in LARG shoot upon exposure
to 10 µM concentration of As(V) & 16 % in HARG shoot which was evident in bound to
free ratio of cysteine (Fig. 4.4). In LARG root the increment was 189% & 42 % in
HARG root at the same treatment of As(V). When treated with 50 µM concentration of
As(V), Cys was increased by 150 % in LARG shoot & by 18 % in HARG shoot. In
LARG root , the increment was 452% and 20 % in HARG root at same concentration of
As(V). Bound to free cysteine was significantly correlated to Metalloid accumulation in
HARG roots upon arsenate (R=0.727***) and arsenite (R=0.705***) exposures
(Appendix C-D).
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Effect of Arsenic Stress on Amino Acid Profile
4.2.1.3. Amino acid status in seeds
The increase in essential amino acids (Fig. 4.5) Thr and Val was 5 times more in
LARG seeds than HARG seeds, while that of Ile and Leu was 2 folds; Phe was 3 folds,
whereas Lys was 23 times more in LARG than HARG (Fig. 4.5). The non-essential
amino acids Asp (8 times), Ser (2 times), Arg (2 times), Ala (1.5 times) and Tyr (6 times)
were found more in LARG seeds than HARG seeds. On the other hand, the amino acids
known to be induced in presence of As were found to be more in HARG seeds viz., Glu
(2 times), Gly (2 times), His (2 times), Pro (1.3 times) and Cys (3 times).
4.3. Discussion
Amino acid profiling of the contrasting genotypes gives us an insight into the
metabolic synapse with metal accumulation and tolerance. The role played by
accumulated free amino acids in plants during heavy metal stress have been previously
investigated (Rai, 2002) but not much concern has been given to functions exhibited by
bound, free amino acids and total amino acids as a whole. In this study we found that
differential As accumulation impacted amino acid profile differentially. Also the ratios
of important known stress induced amino acids Pro and Cys analysed in this study
indicated their importance in stress tolerance and detoxification till a limited As uptake,
henceforth their alterations correspond to plant senescence in HARG.
Amino acid content is depressed by high As exposure (Dwivedi et al., 2010a) as
also are readily peroxidized by the free radicals generated (Gebicki and Gebicki, 1993).
Corresponding to higher As accumulation and lipid peroxidation in HARG most amino
acids showed a sharp decline in presence of As at 25 µM As(III) specially Ile, Leu, Lys,
and Val which, are highly susceptible to free radicals and their subsequent reactions with
protective agents, such as ascorbate or glutathione, decreases the antioxidant potential of
cells and tissues (Gebicki and Gebicki, 1993). Histidine, an important enzyme co-factor
known to accumulate more in heavy metal stress (Sharma and Dietz, 2006), showed a
genotypic specific response (Dwivedi et al., 2010) of a significant increase in LARG
while a decrease in HARG was observed. Proline is known to play a protective role in
plants against active oxygen or hydroxyl radicals (Rai, 2002), further metal tolerant
populations of some plants have been shown to have higher constitutive content of
proline as compared to non-tolerant counterparts (Rai, 2002), corroborating this view
LARG exhibited more constitutive proline content and total proline was also induced
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Effect of Arsenic Stress on Amino Acid Profile
more on As accumulation in LARG than HARG, probably helping in tolerating stress. A
study by Mishra and Dubey (2006) on rice showed enhanced free proline content on
increasing concentrations of arsenite which has also been observed in this study. On the
other hand free to bound ratio of proline was more enhanced in HARG corresponding to
As accumulation suggesting release from protein bound contents. This corresponds with
previous independent observations on free proline and As toxicity that on higher As
accumulation cells signal higher accumulation of stress related metabolites (Maksymiec,
2007) like proline to protect cells from membrane damage (Mishra and Dubey, 2006),
but, when the de-novo synthesis is hampered by arsenite toxicity plant can extract Pro
bound in proteins (Hu et al., 1998) as well.
Cysteine is a central metabolite required in antioxidant defense and metal
detoxification via glutathnione / phytochelatin synthesis (Sharma and Dietz, 2006).
Cysteine synthesis in both the genotypes was positively correlated with As accumulation
but in HARG lesser induction was observed in roots than LARG. The lowering of free to
bound ratio of cysteine with increasing As accumulation in HARG as against a
significant increase in LARG also suggests higher consumption in thiol metabolism but
suppression of de-novo synthesis of cysteine probably due to hindered sulphur uptake
parallel to higher As uptake. Glycine and Glu are also the fundamental metabolites for
phytochelatin and chlorophyll synthesis (Sharma and Dietz, 2006). These were also more
enhanced in LARG shoot as well as root. Further, Glu with Asp are important for other
amino acid synthesis by transamination, while serine participates in ion transport (Rai et
al., 2002). In this study, Asp and Ser decreased to high levels in HARG while
insignificant decrease was observed in LARG. Methionine is the precursor of ethylene,
involved in cell signalling to heavy metal stress (Sharma and Dietz, 2006) and was found
to increase by three folds in LARG whereas, an insignificant increase was observed in
HARG, probably due to increased consumption in ethylene production parallel to higher
As accumulation. Alanine also reported to increase in the presence of As was enhanced
more in LARG (Pavlik et al., 2010).
More As accumulation leads to enhanced H2O2 production which is known to
trigger abscisic acid (ABA) induced stomatal closure, impeding plant growth, but some
amino acids i.e., His, Met and Asp and Glu promote stomatal opening as well and revert
the ABA effect. These amino acids were synthesised more in LARG than HARG.
Overall it can be said that the amino acid profile of HARG hints towards metabolic dysPh.D. thesis / Richa Dave / 2013
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Effect of Arsenic Stress on Amino Acid Profile
regulation and senescence on high As uptake and accumulation either caused by the
metal directly and oxidative stress caused thereby or also due to some signalling
molecules induced by the stress action (Maksymiec, 2007). While in LARG several
stress related amino acids were induced more, leading to enhanced tolerance to arsenic
stress (Schmidt et al., 2005).
4.4. Conclusion
Differential amino acid accumulation response of both the rice genotypes showed
the consequent phytotoxicity or detoxification potential of HARG and LARG
respectively. Several amino acids were affected negatively with high As accumulation,
while some stress responsive amino acids were induced during As exposure. The stress
responsive amino acids proline, cysteine, glycine, glutamic acid and methionine showed
higher accumulation in HARG than LARG. A depressed amino acid profile due to high
As accumulation in HARG was a reflection of phytotoxicity in the genotype.
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